EP2162530B1 - Cellules isolées proliférantes dotées de propriétés de cellules souches de tissus adultes de vertébrés à sang froid, cultures stables de ces cellules et procédé pour leur préparation - Google Patents

Cellules isolées proliférantes dotées de propriétés de cellules souches de tissus adultes de vertébrés à sang froid, cultures stables de ces cellules et procédé pour leur préparation Download PDF

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Publication number
EP2162530B1
EP2162530B1 EP08707810.1A EP08707810A EP2162530B1 EP 2162530 B1 EP2162530 B1 EP 2162530B1 EP 08707810 A EP08707810 A EP 08707810A EP 2162530 B1 EP2162530 B1 EP 2162530B1
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Prior art keywords
cells
tissue
culture
medium
fish
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German (de)
English (en)
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EP2162530A1 (fr
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Charli Kruse
Emel GÜRLEYIK
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Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
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Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening

Definitions

  • the invention relates to a method for producing isolated proliferating cells having stem cell properties and the corresponding stable cell cultures from adult tissue of fish ( pisces ) and the cells and cell cultures produced therewith.
  • Stem cells are those cells that have the ability to divide indefinitely and to differentiate under appropriate circumstances or by appropriate stimuli in different cell types. Stem cells have the potential to develop into cells with distinctive shape and specialized functions. These specialized cells are often very limited or not cultivated outside of the organism, so they must be produced via the detour of the culture of stem cells.
  • Embryonic stem cells have so far been isolated and studied by a few fish species (zebrafish and medaka) [ Ma et al., 2001, Proc. Natl. Acad. Sci. USA 98: 2461-2466 ; Epub 2001, Feb. 13; Hong et al., 1996, Mech. Develop., 60: 33-44 ], whereas only very few studies are known about adult stem cells from fish [ Tawk and Vriz 2003, Med. Sci. (Paris) 19: 465-471 ; Alvarado and Tsonis al., 2006, Nat. Rev. Genet., 7: 873-884 ]. However, these studies were performed on complex fish tissues, usually in vivo, on the fin, heart and brain, and did not allow the isolation of isolated stem cells or stable stem cell cultures.
  • the invention is therefore based on the object to obtain isolated proliferating cells with stem cell properties, in particular unlimited ability to divide and multipotency or pluripotency, from adult tissue of fish and establish stable proliferating cell cultures thereof, with which a wide range of different differentiated cells can be produced.
  • the fish is in particular a Nutzfisch.
  • suitable species are sturgeon, herring, trout, salmon, eel, carp.
  • kidney tissue As an adult starting tissue, kidney tissue is used.
  • the head kidney has blood-forming functions in fish.
  • the mammalian hematopoietic stem cells are known to dissociate from the substrate after differentiation into mature blood cells under cell culture conditions.
  • the cells prepared according to the invention were dissected from fish head kidneys Not even after more than 18 passages were still clinging to the ground and exhibiting characteristics of stem cells similar to those isolated from exocrine glands. This result was not expected.
  • the proliferating cells provided by the invention have stem cell properties, that is, they have unrestricted ability to divide, and moreover are multipotent or pluripotent, that is, they can spontaneously differentiate into cells of at least two cotyledons (ectoderm and mesoderm).
  • the simple method according to the invention makes it possible to produce adult stem cells which, in their differentiation potential, reflect different cotyledons and can therefore at least be termed multipotent.
  • the stem cells produced by the method according to the invention are easy to obtain and reproducibly differentiate spontaneously into different cell types. So far, muscle cells, glial cells, epithelial and endothelial cells have been detected, but the available cell types are certainly not limited to this and there are already findings that indicate other cell types.
  • feeder cells includes all cells which promote the growth of the cells actually to be cultivated by releasing growth factors and / or providing an extracellular matrix or preventing the differentiation of the stem cell culture.
  • stem cells produced according to the invention can proliferate in adhesion culture.
  • conventional hematopoietic stem cells are usually grown in suspension culture.
  • stem cell cultures of the inventors after more than 25 passages over 6 months, the cells have retained their ability to self-renew and unrestricted division, and the cultures remain stable.
  • stem cells obtained according to the invention can be stored frozen (preferably in liquid nitrogen) and retain their differentiability and vitality unchanged.
  • the cells and cell cultures according to the invention can already be cultivated at temperatures lower than 37 ° C. (typical for mammalian cell cultures).
  • the suitable temperature range will depend on the nature and source of the fish species used as the tissue source, and thus may include a broad spectrum of about 1-35 ° C.
  • a suitable cultivation temperature may be in the upper range, e.g. about 25-35 ° C, while for cells of fish from cold regions e.g. 1-10 ° C can be.
  • the temperature range will typically be in a range of 5-27 ° C, preferably 10-25 ° C, more preferably 17-24 ° C, especially 18-20 ° C or about room temperature.
  • the respective suitable or optimum temperature range can easily be determined by the skilled person by means of routine tests.
  • the stem cells and stem cell cultures produced according to the invention have a wide spectrum of applications.
  • these cellular systems comprise several types of cells, e.g. those that occur side by side in natural tissues, which can also be tested at the same time.
  • the stem cells and stem cell cultures produced according to the invention can also be used to develop tissue-like or organ-like multicellular systems, preferably from several cell types, in vitro .
  • these can be used for food and feed production. They may be used, for example, to produce fish-specific biomass, e.g. artificial meat, fishmeal, fish oil or individual components of these cells, e.g. Omega fatty acids or proteins with an amino acid composition typical for fish.
  • fish-specific biomass e.g. artificial meat, fishmeal, fish oil or individual components of these cells, e.g. Omega fatty acids or proteins with an amino acid composition typical for fish.
  • These substances may be certain substances that are naturally produced in the parent organisms, eg fish.
  • a specific example of this are special proteins, eg cold-adapted enzymes with an optimum activity at low temperatures (eg about 10 ° C), including applications in food ripening.
  • Such enzymes could, for example, be produced by glandular cells which are formed by spontaneous or targeted differentiation from the stem cells according to the invention.
  • the stem cells and cell cultures prepared according to the invention can also be used as recipient cells or vehicles for receiving foreign genetic material or other foreign material according to methods basically known in the art.
  • the expression of the foreign gene material then allows the production of heterogeneous proteins in these cells and cultures.
  • a particular advantage of such cells and cell cultures is that they are already at temperatures lower than 37 ° C (typical for mammalian cell cultures), e.g. in a range of 1-35 ° C, preferably 5-27 ° C or 17-24 ° C, especially 18-20 ° C or about room temperature, grow well, which makes energy-intensive heating unnecessary.
  • Yet another possible application of the cells according to the invention is the reproductive cloning of vertebrate animals, especially for fish farming (clone fish).
  • An embodiment of the present invention also relates to a kit comprising the cells according to any one of claims 4 to 6 and further adjuvants or reagents, e.g. Reagents for culturing the cells or diagnostic reagents.
  • adjuvants or reagents e.g. Reagents for culturing the cells or diagnostic reagents.
  • the cytokeratin structural proteins are mainly formed in epithelial and endothelial cells.
  • the protein Vigilin is typical of protein-synthesizing cells and has been found in all stem cells so far.
  • Fig. 1 Scheme shown is used to recover the cells of the kidney tissue mechanically and enzymatically crushed in culture.
  • no complete tissue blocks are taken into culture, but intact cell aggregates, which are smaller than about 200 microns, placed in culture vessels from which then grow the sought cells or re-form.
  • HEPES stock solution pH 7.6
  • HEPES Eagle medium 90 ml Modified Eagle Medium (MEM)
  • MEM Modified Eagle Medium
  • Isolation medium pH 7.4
  • 32 ml of HEPES Eagle's medium 8 ml of 5% BSA in A. bidest.
  • DMEM Dulbecco's Modified Eagle Medium
  • FCS or fungal or algae extracts
  • the head kidney of a sturgeon is removed from the surface of the upper body cavity just behind the skull starting. Thereafter, the tissue is minced in a beaker with digestion medium with a fine pair of scissors. The suspension is then gassed for 1 min with carbogen and incubated for 20 min at 37 ° C in a shaker at 200 cycles / min. Then the medium is carefully aspirated off and the tissue pieces are washed twice with 10 ml of isolation medium each time and 5-10 ml of digestion medium are again added to the tissue.
  • tissue pieces are crushed by successively pulling in each of a 10 ml, 5ml and 2 ml glass pipette and through single-layer filter fabric with a mesh size pressed by about 200 ⁇ m.
  • the cells thus separated are then washed 1-2 times in incubation medium (37 ° C) and centrifuged each time for 5 min at 90 g (800 rpm). The final pellet is resuspended in incubation medium and on tissue culture dishes (25 cm 2) distributed.
  • tissue culture dishes with the isolated cells are cultured in the incubator at 18-20 ° C and 5% CO 2 . Every 3-4 days, the medium is changed. This removes all non-adherent and many differentiated cells.
  • the cells After the cells completely cover the soil (ie are confluent), for the first time on the 10th day of culture, the cells are passaged with a solution consisting of 2 ml PBS, 1 ml trypsin and 2 ml incubation medium at 37 ° C. Solve it the cells from the bottom of the culture dish. The cell suspension is centrifuged for 5 minutes, the supernatant aspirated and the cells resuspended in 5 ml of incubation medium, transferred to a medium cell culture flask (75 cm 2 ) and 10 ml of incubation medium added. The media change takes place every three days.
  • the cells are re-passaged, but this time with 6 ml PBS, 2 ml trypsin and 6 ml incubation medium.
  • the cell suspension is centrifuged for 5 minutes, the supernatant is aspirated off and the cells are resuspended in 15 ml of incubation medium, transferred to 3 medium cell culture flasks and 10 ml each of incubation medium added.
  • the cells are further cultured and passaged and seeded until the cells reach a semi-confluent to confluent condition.
  • the cells may be transferred to differentiation medium (e.g., with the composition given in the table above) and differentiated under known culture conditions.
  • differentiation medium e.g., with the composition given in the table above
  • additional differentiation and growth factors are not necessarily required because the cells of the invention can spontaneously differentiate into different cell types. Nevertheless, to promote targeted differentiation into certain cell types, it may be advantageous to add such differentiation and growth factors.
  • Such factors that specifically promote the differentiation of stem cells into particular cell types are known in the art for a variety of cell types and can be used analogously to known protocols.

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  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
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  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Claims (14)

  1. Procédé servant à fabriquer des cellules adultes proliférantes présentant des propriétés de cellules souches, qui peuvent être différenciées au moins en cellules des deux foyers embryonnaires ectoderme et mésoderme, comprenant les étapes consistant à :
    prélever du tissu du rein antérieur d'un poisson,
    fractionner le tissu prélevé,
    cultiver le tissu fragmenté, et
    multiplier les cellules persistantes en culture,
    dans lequel le tissu est fragmenté avec ménagement de sorte que des amas cellulaires qui sont inférieurs à environ 200 µm sont largement préservés dans les fragments de tissus résultant,
    et le tissu fractionné est cultivé en une culture adhérente et les cellules adhérentes persistantes en culture continuent à être cultivées et les cellules différenciées non adhérentes sont séparées par un changement de milieu.
  2. Procédé selon la revendication 1, caractérisé en ce que le tissu fractionné est cultivé dans un premier temps dans des conditions adaptées dans des récipients de culture de tissu, dans lequel la majeure partie des cellules différenciées meurt rapidement en quelques jours et se détache des cellules souches, les cellules souches se fixant après quoi au niveau du fond du récipient de culture de tissu,
    et le tissu restant et des cellules différenciées non-adhérentes sont largement séparés par un premier changement de milieu, et
    des cellules non-adhérentes restantes sont séparées par d'autres changements de milieux à des intervalles de quelques jours, de préférence d'environ 2 - 3 jours.
  3. Procédé selon l'une quelconque des revendications 1 à 2, caractérisé en ce que la mise en culture et la multiplication des cellules persistantes en culture sont effectuées à une température de 5-27 °C, en particulier de 17-24 °C.
  4. Cellules proliférantes présentant des propriétés de cellules souches, pouvant être obtenues à l'aide du procédé selon l'une quelconque des revendications 1-3 à partir du tissu de rein antérieur d'un poisson, caractérisées en ce que les cellules présentent une capacité de division illimitée et peuvent être différenciées au moins en des cellules des deux foyers embryonnaires, ectoderme et mésoderme.
  5. Cellules selon la revendication 4, caractérisées en ce que les cellules conservent également après la congélation/cryoconservation leur capacité d'autorenouvellement et de division illimitée et ne sont pas différenciées.
  6. Cellules selon la revendication 4 ou 5, caractérisées en ce que les cellules présentent l'aptitude à la prolifération en culture d'adhésion.
  7. Culture de cellules, en particulier culture d'adhésion, comprenant les cellules selon l'une quelconque des revendications 4 à 6 dans un milieu de culture, qui permet la conservation stable et la multiplication des cellules sensiblement sans différenciation.
  8. Culture de cellules selon la revendication 7, caractérisée en ce que les cellules conservent leur capacité à l'autorenouvellement et à la division illimitée pour plus de 25 passages.
  9. Culture de différenciation, comprenant les cellules selon l'une quelconque des revendications 4 à 6 ainsi que des cellules différenciées en résultant dans un milieu de différenciation, caractérisée en ce que le milieu de différenciation ne comprend aucun facteur de croissance ou de différenciation supplémentaire.
  10. Utilisation des cellules selon l'une quelconque des revendications 4 à 6 ou de la culture de cellules selon l'une quelconque des revendications 7 à 8 servant à développer des systèmes multicellulaires similaires à des tissus ou similaires à des organes in vitro.
  11. Utilisation des cellules selon l'une quelconque des revendications 4 à 6 ou de la culture de cellules selon l'une quelconque des revendications 7 à 8 en tant que système in vitro pour tester des produits chimiques, en particulier pour cribler des principes actifs médicamenteux, ou en tant que système in vitro pour la production de substances souhaitées in vitro.
  12. Utilisation selon la revendication 11, caractérisée en ce que des protéines, en particulier des enzymes présentant une activité optimale à des températures basses, des huiles de poisson ou des acides gras sont produits.
  13. Utilisation des cellules selon l'une quelconque des revendications 4 à 6 ou de la culture de cellules selon l'une quelconque des revendications 7 à 8 pour le clonage reproductif de vertébrés à sang froid, en particulier pour l'élevage de poissons, ou en tant que cellules réceptrices ou véhicules servant à recevoir un matériel génétique étranger ou d'autre matériel étranger.
  14. Jeu, comprenant les cellules selon l'une quelconque des revendications 4 à 6, ainsi que d'autres agents auxiliaires ou réactifs, par exemple des réactifs servant à la mise en culture des cellules ou des réactifs de diagnostic.
EP08707810.1A 2007-06-27 2008-02-27 Cellules isolées proliférantes dotées de propriétés de cellules souches de tissus adultes de vertébrés à sang froid, cultures stables de ces cellules et procédé pour leur préparation Active EP2162530B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102007029699A DE102007029699B4 (de) 2007-06-27 2007-06-27 Isolierte proliferierende Zellen mit Stammzelleigenschaften aus adultem Gewebe von wechselwarmen Wirbeltieren, stabile Zellkulturen davon und Verfahren zu deren Herstellung
PCT/EP2008/001550 WO2009000347A1 (fr) 2007-06-27 2008-02-27 Cellules isolées proliférantes dotées de propriétés de cellules souches de tissus adultes de vertébrés à sang froid, cultures stables de ces cellules et procédé pour leur préparation

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EP2162530A1 EP2162530A1 (fr) 2010-03-17
EP2162530B1 true EP2162530B1 (fr) 2017-06-07

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US (1) US20100319079A1 (fr)
EP (1) EP2162530B1 (fr)
JP (1) JP5301536B2 (fr)
DE (1) DE102007029699B4 (fr)
WO (1) WO2009000347A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2500412B8 (fr) 2011-03-15 2015-02-25 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Procédé de production de cellules de poisson ayant une teneur plus élevée en acides gras hautement insaturés et utilisation de ces cellules de poisson pour la production de produits spécifiques aux poissons
CN111154726A (zh) 2013-10-30 2020-05-15 密苏里大学的策展人 可扩展性骨骼肌谱系特化和培育的方法
WO2018208628A1 (fr) 2017-05-06 2018-11-15 Memphis Meats, Inc. Compositions et procédés permettant d'augmenter la densité de culture d'une biomasse cellulaire dans une infrastructure de culture
US11479792B2 (en) 2017-07-13 2022-10-25 Upside Foods, Inc. Compositions and methods for increasing the efficiency of cell cultures used for food production

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ANDERSON M J ET AL: "Molecular differentiation of neurons from ependyma-derived cells in tissue cultures of regenerating teleost spinal cord", MOLECULAR BRAIN RESEARCH, ELSEVIER SCIENCE BV, AMSTERDAM, NL, vol. 2, no. 2, 1 July 1987 (1987-07-01), pages 131 - 136, XP025435654, ISSN: 0169-328X, [retrieved on 19870701], DOI: 10.1016/0169-328X(87)90006-4 *

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JP5301536B2 (ja) 2013-09-25
US20100319079A1 (en) 2010-12-16
JP2010531146A (ja) 2010-09-24
WO2009000347A1 (fr) 2008-12-31
DE102007029699B4 (de) 2010-12-02
DE102007029699A1 (de) 2009-01-02
EP2162530A1 (fr) 2010-03-17

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